Project Summary/Abstract Candidate Jeffrey O. Bush is a postdoctoral fellow who received his Ph.D. at the University of Rochester for work in understanding cleft lip and palate in the spontaneously-occurring Dancer mutant mouse. This work identified gain of function of the transcription factor Tbx10 as being causative to the cleft lip and palate phenotype in the Dancer mice and identified one of a very few known genetic causes for this phenotype in mice. As a postdoctoral fellow in the laboratory of Philippe Soriano, the candidate has focused on understanding ephrin-B1 function in the etiology of a congenital disorder affecting craniofacial development, Craniofrontonasal syndrome (CFNS). This work has shown that the cleft palate phenotype associated with ephrin-B1 loss of function is caused by defective anterior palatal shelf outgrowth and proliferation, as a consequence of loss of forward signaling. The candidate's short-term career goal is to evolve these studies into an independent research program studying Eph/ephrin signaling in craniofacial development, with a long- term career goal to expand this research program to study other craniofacial congenital defects with a focus on signaling molecules involved in craniofacial development and disease. Environment The proposed work will take place in the laboratory of Professor Philippe Soriano in the Department of Developmental and Regenerative Biology at the Mount Sinai School of Medicine. The laboratory has a world- class record of accomplishment in mouse genetics studies of signaling function during development, and has made significant contribution into the understanding of Platelet-Derived Growth Factor signaling throughout development, including craniofacial development. The Department of Developmental and Regenerative Biology includes fourteen full-time faculty that study various questions centered on the development, regeneration, and patterning of organs. The resources available within the laboratory and the department will provide significant support to the candidate during the mentored phase and transition to independence. Research Craniofacial malformations are extremely common, identified in three quarters of all congenital abnormalities identified at birth. These include cleft palate, a failure of the roof of the mouth to join at birth which occurs in approximately 1 in 1000 live births. The treatment of craniofacial conditions involves multiple invasive surgeries, and has a dramatic impact on an affected individual's childhood health and family. One such genetic disorder, Craniofrontonasal syndrome (CFNS) is caused by mutations in the ephrin-B1 gene. This is an X-linked disorder that causes a number of craniofacial defects including, hypertelorism, nasal grooves, coronal craniosynostosis and cleft lip and palate. Mutations in the same gene in mice cause the same defects, supporting the idea that the mouse is a good model organism for studying this and other craniofacial diseases. Ephrin-B1 is a member of a family of signaling molecules that act by activating EphB receptors. This family of genes have important functions in a wide variety of developmental and disease contexts. We have data indicating that an additional ephrin gene, ephrin-B2, may play important roles in craniofacial development and disease. In the first aim of this application, I propose to study ephrin-B2 function in craniofacial development by studying mice in which its function is removed from specific craniofacial tissues during development. I will do this by utilizing currently available alleles to remove ephrin-B2 from the palatal shelf epithelium, where it is highly expressed. Additionally, I will take a tissue-specific rescue strategy to test for requirement for ephrin-B2 during branchial arch formation. Based on preliminary expression data, EphB4, an important receptor for ephrin-B2 is highly expressed within the palate during its formation. In the second aim, I therefore propose to test for a role of EphB4 during formation of the secondary palate by generating a conditional allele to perform tissue-specific disruption of EphB4 function in the neural crest-derived mesenchyme. Finally, I have initiated studies to identify downstream components of the Eph/ephrin signaling network by developing a mass-spectrometry based proteomic approach to identify phosphorylation targets of EphB/ephrin-B signaling in the palate. This approach has already identified a large number of excellent candidate molecules for transduction of downstream signaling. These candidates have mostly unknown roles in craniofacial development, and I therefore propose in the third aim to prioritize and study these candidates, with the goal of elaborating signaling pathways downstream of ephrin-B signaling that control palate formation. This study will greatly enhance understanding of the genetic causes of cleft palate by identifying the importance of ephrin-B2 and EphB4 in its development, and by characterizing new genes with previously unknown importance in craniofacial development and disease.